【作者】 叶意群；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2006， 硕士

【摘要】 本实验室在前一阶段利用cDNA-AFLP技术结合RACE技术,从白菜(Brassica campestris L.ssp.chinensis Makino)核雄性不育两用系中分离得到了一个与白菜核雄性不育相关的编码多聚半乳糖醛酸酶的基因BcMF9。多聚半乳糖醛酸酶的多基因家族成员在植物发育不同阶段和不同组织中表达,它们与果实成熟、细胞分离过程如果实软化、叶和花的脱落、花粉成熟等都有关。我们在前期利用RT-PCR和Northern杂交验证都表明BcMF9基因是在花粉中特异表达,而在叶片和茎等孢子体组织中不表达(数据未发表),表明其可能是一个在花粉发育中扮演重要角色的基因。为进一步验证该基因的的生物学功能,我们对BcMF9进行了核苷酸和蛋白质序列分析,并在此基础上分别构建了CaMV35S组成型表达载体pBI35S-BcMF9A和BcA9绒毡层组织特异型植物表达载体pBIA9-BcMF9A采用反义技术将其导入正常可育菜心(B.campestris ssp.chinensis var. parachinensis Tsen et Lee)中,继而,对转基因植株进行分子水平的检测,及花粉形态观察、花粉萌发能力的测定等。主要结果如下: (1) BcMF9基因DNA全长1481bp,cDNA序列全长为1286 bp利用DNAStar软件对BcMF9基因序列进行分析,发现其最大开放阅读框为1188 bp,编码395个氨基酸。BLAST搜索发现BcMF9基因cDNA全序列与拟南芥多聚半乳糖醛酸酶PGA3 mRNA序列相似性最高,为88%(1023/1161)。采用MEGA3.1软件对BcMF9与GenBank数据库中其它植物PG基因的CDS序列构建系统树,发现系统树可明显分为3个分支,BcMF9基因聚在分支C上,有研究和数据显示,这一分支上的其它基因均在花粉或花蕾中表达,初步表明BcMF9基因可能是花粉特异表达的多聚半乳糖醛酸酶基因。 (2) 构建了反义BcMF9基因的CaMV35S组成型表达载体pBI35S-BcMF9A和BcA9绒毡层组织特异型表达载体pBIA9-BcMF9A,并成功导入农杆菌LBA4404菌株中。 (3) 按本实验室已建立的白菜高效遗传转化体系,获得了12个抗卡那霉素的pBI35S-BcMF9A转基因芽系和10个pBIA9-BcMF9A转基因芽系,100多株抗性苗。对这些转基因植株和非转化菜心植株分别提取DNA和RNA,经PCR检测,结果表明转化的阳性率为90.56%。随后以转基因‘油青’菜心的对照株为阴性对照进行X-Gluc组织化学染色检测,结果显示pBI35S-BcMF9A转基因‘油青’菜心植株的叶边缘被染上了蓝色,而非转基因植株没有蓝色斑点,更多还原

【Abstract】 A polygalacturonase (PG) gene BcMF9 was obtained in floral bud of Chinese cabbage pak-choi (B. campestris ssp. chinensis (L.) Makino, syn. B. rapa ssp. chinensis (L.) Makino) between A line and B line by cDNA-AFLP technology and rapid amplification of cDNA ends (RACE) technology. The multi gene family of PG expressed in the different development stages and different tissues of plants, such as fruit softening,pollen maturation and so on, which related to fruit ripen, cell separation and others. The result of Northern hybridization and RT-PCR confirmed that gene BcMF9 was inhibited obviously in the male sterile line, and the gene only expressed in floral bud but not in leaf and stem. These results shows that BcMF9 may expressed in pollen development. In order to find out the function of gene BcMF9 in detail, we analyse the sequence of BcMF9 and construct the plant expressing plasmid vector and the explants were inoculated with Agrobacterium tumefaciens strain LBA4404 containing pBI35S-BcMF9A or pBIA9-BcMF9A carrying antisense gene BcMF9. Then, the expressing characteristics of gene BcMF9 were analyzed by Northern and Southern hybridization in transgenic plantlets, non-transgenic plantlets of the genic male sterile AB line. At the same time, in order to understand the biological function of gene BcMF9, morphology and the development course of microsporogenesis were observed in transgenic plantlets and non-transgenic plantlets. The primary results obtained from the research provide some clues for the study on the biological function of gene BcMF9A and molecular mechanism of the male sterility. The main results are as follow:(1) The largest open reading frame of BcMF9 is 1188 bp and encodes a protein of 395 amino acids with the DNA sequence of BcMF9 is 1481bp, full-length cDNA is 1286 bp.it interrupted by two introns of 32 bp, 129 bp. The similarity of BcMF9 and PGA3, a PG gene related to plant pollen development in Arabidopsis thaliana is 88%. Phylogenetic analysis showed that BcMF9 falls into the category of clade-C, which includes PG related to pollen. These results test that BcMF9 may acts as a pollen-specific polygalacturonase.(2) After the constitutive type plant expressing plasmid vector (pBI35S-BcMF9A) and the tapetum-spccific plant expressing plasmid vector (pBIA9-BcMF9A) were constructed, we introduced them into Agrobacterium tumefaciens strain LBA4404.(3) More than 100 plantlets Kan~R seedlings are obtained based on the efficient genetic transformation system which have been obtained in our lab. PCR, Southern blotting, Northern blottingand GUS expression staining method of histochemistry and pollen germinateion have done. The frequency of positive to PCR is 90.56%. The result of GUS expression staining method of histochemistry show that there are GUS expression in the pBI35S-BcMF9A transgenic plantlets leaves and in the pB!A9-BcMF9A transgenic plantlets anther, but none in the non-transgenic plantlets. The Southern blotting show the antisense BcMF9 gene was integrated into flowering Chinese cabbage genome.(4) The result of Northern hybridization indicated that antisense gene BcMF9 was inhibited obviously in the transgenic plantlets, and it confirmed that antisense gene BcMF9 has been expressed in flowering Chinese cabbage, and the antisense gene BcMF9 fragment of the transgenic plant has been transcripted actually to inhibit the normal expression of gene BcMF9.lt hints that the antisense gene BcMF9 were transcripted and expressed correctly. Only 30% ~ 40% pollen germination in the transgenic plantlets,but more than 90% in the non-transgenic plantlets. It shows that pollen germination of transgenic plantlets were disturbed. These results predicted that BcMF9 gene was related to the pollen development, whether it expressed effected the normal germination of pollen.更多还原